Tissue Compartments, Generally
In multicellular organisms, cells that are specialized to perform common functions are usually organized into cooperative assemblies embedded in a complex network of secreted extracellular macromolecules, the extracellular matrix (ECM), to form specialized tissue compartments. Individual cells in such tissue compartments are in contact with ECM macromolecules. The ECM helps hold the cells and compartments together and provides an organized lattice or construct within which cells can migrate and interact with one another. In many cases, cells in a compartment can be held in place by direct cell-cell adhesion. In vertebrates, such compartments may be of four major types, a connective tissue (CT) compartment, an epithelial tissue (ET) compartment, a muscle tissue (MT) compartment and a nervous tissue (NT) compartment, which are derived from three embryonic germ layers: ectoderm, mesoderm and endoderm. The NT and portions of the ET compartments are differentiated from the ectoderm; the CT, MT and certain portions of the ET compartments are derived from the mesoderm; and further portions of the ET compartment are derived from the endoderm.
The Bone Marrow Niche
The term “niche” as used herein refers to a specialized regulatory microenvironment, consisting of components which control the fate specification of stem and progenitor cells, as well as maintaining their development by supplying the requisite factors. The term “bone marrow (BM) niche” as used herein refers to a well-organized architecture composed of osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells, adipocytes and extracellular matrix proteins (ECM). These elements play an essential role in the survival, growth and differentiation of diverse lineages of blood cells.
Bone marrow consists of a variety of precursor and mature cell types, including hematopoietic cells (the precursors of mature blood cells) and stromal cells (the precursors of a broad spectrum of connective tissue cells), both of which appear to be capable of differentiating into other cell types. The mononuclear fraction of bone marrow contains stromal cells, hematopoietic precursors, and endothelial precursors.
Extracellular Matrix (ECM) Proteins
The ECM is a complex structural entity surrounding and supporting cells found within mammalian tissues. The ECM is comprised of proteoglycans (e.g., heparan sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid), collagen, fibronectin, laminin and elastin. Most mammalian cells cannot survive unless they are anchored to the ECM. Cells attach to the ECM via transmembrane glycoproteins (e.g., integrins) which bind to various types of ECM proteins (e.g., collagens, laminins, fibronectin).
Adipocytes
Adipocytes of the bone marrow stroma provide the cytokines and extracellular matrix proteins required for the maturation and proliferation of the circulating blood cells. Due to the complexity of the bone marrow as an organ, the normal physiology of these stromal cells is not well understood. In particular, the role of adipocytes in the bone marrow remains controversial. Cloned bone marrow stromal cell lines provide an in vitro model for analysis of the lympho-hematopoietic microenvironment. These cells may be capable of multiple differentiation pathways, assuming the phenotype of adipocytes, chondrocytes, myocytes, and osteocytes in vitro. (Gimble J M, New Biol., 1990 April; 2(4): 304-312).
Hematopoietic Stem Cells Development and Maintenance
Hematopoietic stem cells (HSCs) (also known as the colony-forming unit of the myeloid and lymphoid cells (CFU-M,L), or CD34+ cells) are rare pluripotential cells within the blood-forming organs that are responsible for the continued production of blood cells during life. While there is no single cell surface marker exclusively expressed by hematopoietic stem cells, it generally has been accepted that human HSCs have the following antigenic profile: CD 34+, CD59+, Thy1+ (CD90), CD38low/−, C-kit−/low and, lin− (Chotinantakul, K. and Leeanansaksiri, W., Bone Marrow Research, Vol. 2012, Article ID 270425; The National Institutes of Health, Resource for Stem Cell Research, http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx). CD45 is also a common marker of HSCs, except platelets and red blood cells (The National Institutes of Health, Resource for Stem Cell Research, http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx). HSCs can generate a variety of cell types, including erythrocytes, neutrophils, basophils, eosinophils, platelets, mast cells, monocytes, tissue macrophages, osteoclasts, and the T and B lymphocytes (The National Institutes of Health, Resource for Stem Cell Research, http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx). The regulation of hematopoietic stem cells is a complex process involving self-renewal, survival and proliferation, lineage commitment and differentiation and is coordinated by diverse mechanisms including intrinsic cellular programming and external stimuli, such as adhesive interactions with the microenvironmental stroma and the actions of cytokines (Chotinantakul, K. and Leeanansaksiri, W., Bone Marrow Research, Vol. 2012, Article ID 270425; The National Institutes of Health, Resource for Stem Cell Research, http://stemcells.nih.gov/info/scireport/pages/chapter5.aspx).
Different paracrine factors are important in causing hematopoietic stem cells to differentiate along particular pathways. Paracrine factors involved in blood cell and lymphocyte formation are called cytokines. Cytokines can be made by several cell types, but they are collected and concentrated by the extracellular matrix of the stromal (mesenchymal) cells at the sites of hematopoiesis (Majumdar, M. K. et al., J. Hematother. Stem Cell Res. 2000 December; 9(6): 841-848). For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) and the multilineage growth factor IL-3 both bind to the heparan sulfate glycosaminoglycan of the bone marrow stroma (Burdon, T. J., et al., Bone Marrow Research, Volume 2011, Article ID 207326; Baraniak, P. R. and McDevitt, T. C., Regen. Med. 2010 January; 5(1): 121-143). The extracellular matrix then presents these factors to the stem cells in concentrations high enough to bind to their receptors.
Mesenchymal Stem Cells (MSCs)
Mesenchymal stem cells (MSCs) (also known as bone marrow stromal stem cells or skeletal stem cells) are non-blood adult stem cells found in a variety of tissues. They are characterized by their spindle-shape morphologically; by the expression of specific markers on their cell surface; and by their ability, under appropriate conditions, to differentiates along a minimum of three lineages (osteogenic, chondrogenic, and adipogenic) (Minguell, J. J., et al., Experimental Biology and Medicine 2001, 226: 507-520; Tuan, R. S., et al., Arthritis Res. Ther. DOI: 10.1186/ar614).
No single marker that definitely delineates MSCs in vivo has been identified due to the lack of consensus regarding the MSC phenotype, but it generally is considered that MSCs are positive for cell surface markers CD105, CD166, CD90, and CD44 and that MSCs are negative for typical hematopoietic antigens, such as CD45, CD34, and CD14 (Minguell, J. J., et al., Experimental Biology and Medicine 2001, 226: 507-520; Lee, H. J., et al., Arthritis & Rheumatism, Vol. 60, No. 8, August 2009, pp. 2325-2332; Kolf, C. M., et al., Arthritis Research & Therapy 2007, 9:204, DOI: 10.1186/ar2116). As for the differentiation potential of MSCs, studies have reported that populations of bone marrow-derived MSCs have the capacity to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic-supporting stroma (Gimbel, J. M., et al., Transfus. Med. Hemother. 2008; 35: 228-238; Minguell, J. J., et al., Experimental Biology and Medicine 2001, 226: 507-520; Kolf, C. M., et al., Arthritis Research & Therapy 2007, 9:204, DOI: 10.1186/ar2116). Studies using transgenic and knockout mice and human musculoskeletal disorders have reported that MSC differentiate into multiple lineages during embryonic development and adult homeostasis (Komine, A., et al., Biochem. Biophys. Res. Commun. 2012 Oct. 5; 426(4): 468-474; Shen, J., et al., Scientific Reports, 1:67, DOI: 10.1038/srep00067; Reiser, J., et al., Expert. Opin. Biol. Ther. 2005 December; 5(12): 1571-1584).
Analyses of the in vitro differentiation of MSCs under appropriate conditions that recapitulate the in vivo process have led to the identification of various factors essential for stem cell commitment. Among them, secreted molecules and their receptors (e.g., transforming growth factor-β), extracellular matrix molecules (e.g., collagens and proteoglycans), the actin cytoskeleton, and intracellular transcription factors (e.g., Cbfa1/Runx2, PPAR, Sox9, and MEF2) have been shown to play important roles in driving the commitment of multipotent MSCs into specific lineages, and maintaining their differentiated phenotypes (Kolf, C. M., et al., Arthritis Research & Therapy 2007, 9:204, DOI: 10.1186/ar2116).
For example, it has been shown that osteogenesis of MSCs, both in vitro and in vivo, involves multiple steps and the expression of various regulatory factors. During osteogenesis, multipotent MSCs undergo asymmetric division and generate osteoprecursors, which then progress to form osteoprogenitors, preosteoblasts, functional osteoblasts, and eventually osteocytes (Bennett, K. P., et al., BMC Genomics 2007, 8:380, DOI: 10.1186/1471-2164-8-380). This progression from one differentiation stage to the next is accompanied by the activation and subsequent inactivation of transcription factors, i.e., Cbfa1/Runx2, Msx2, Dlx5, Osx, and expression of bone-related marker genes, i.e., osteopontin, collagen type I, alkaline phosphatase, bone sialoprotein, and osteocalcin (Bennett, K. P., et al., BMC Genomics 2007, 8:380, DOI: 10.1186/1471-2164-8-380, Ryoo, H. M., et al., Mol. Endo. 1997, Vol. 11, No. 11, pp. 1681-1694; Hou, Z. et al., Proc. Natl. Acad. Sci., Vol. 96, pp. 7294-7299, June 1999; Engler, A. J., et al., Cell 126, 677-689, Aug. 25, 2006; Marom, R. et al., Journal of Cellular Physiology 202: 41-48 (2005)). Members of the Wnt family also have been shown to impact MSC osteogenesis. Wnts are a family of secreted cysteine-rich glycoproteins that have been implicated in the regulation of stem cell maintenance, proliferation, and differentiation during embryonic development. Canonical Wnt signaling increases the stability of cytoplasmic β-catenin by receptor-mediated inactivation of GSK-3 kinase activity and promotes β-catenin translocation into the nucleus (Liu, G., et al., JCB, Vol. 185, No. 1, 2009, pp. 67-75). The active β-catenin/TCF/LEF complex then regulates the transcription of genes involved in cell proliferation (Novak, A. and Dedhar, S., Cell. Mol. Life Sci. 1999 Oct. 30; 56(5-6); 523-537; Grove, E. A., Genes and Development 2011 25: 1759-1762). In humans, mutations in the Wnt co-receptor, LRP5, lead to defective bone formation (Krishnan, V., et al., The Journal of Clinical Investigation, Vol. 116, No. 5, May 2006, pp. 1202-1209). “Gain of function” mutation results in high bone mass, whereas “loss of function” causes an overall loss of bone mass and strength, indicating that Wnt signaling is positively involved in embryonic osteogenesis (Krishnan, V., et al., The Journal of Clinical Investigation, Vol. 116, No. 5, May 2006, pp. 1202-1209; Niziolek, P. J., et al., Bone 2011 November; 49(5): 1010-1019). Canonical Wnt signaling pathway also functions as a stem cell mitogen via stabilization of intracellular β-catenin and activation of the β-catenin/TCF/LEF transcription complex, resulting in activated expression of cell cycle regulatory genes, such as Myc, cyclin D1, and Msx1 (Willert, J., et al., BMC Development Biology 2002, 2:8, pp. 1-7). When MSCs are exposed to Wnt3a, a prototypic canonical Wnt signal, under standard growth medium conditions, they show markedly increased cell proliferation and a decrease in apoptosis, consistent with the mitogenic role of Wnts in hematopoietic stem cells (Almeida, M., et al., The Journal of Biological Chemistry, Vol. 280, No. 50, pp. 41342-41351, Dec. 16, 2005; Vijayaragavan, K., et al., Cell Stem Cell 4, 248-262, Mar. 6, 2009). However, exposure of MSCs to Wnt3a conditioned medium or overexpression of ectopic Wnt3a during osteogenic differentiation inhibits osteogenesis in vitro through β-catenin mediated down-regulation of TCF activity (Quarto, N., et al., Tissue Engineering: Part A, Vol. 16, No. 10, 2010, pp. 3185-3197). The expression of several osteoblast specific genes, e.g., alkaline phosphatase, bone sialoprotein, and osteocalcin, is dramatically reduced, while the expression of Cbfa1/Runx2, an early osteo-inductive transcription factor is not altered, implying that Wnt3a-mediated canonical signaling pathway is necessary, but not sufficient, to completely block MSC osteogenesis (Quarto, N., et al., Tissue Engineering: Part A, Vol. 16, No. 10, 2010, pp. 3185-3197; Eslaminejad, M. B. and Yazdi, P. E., Yakhteh Medical Journal, Vol. 9, No. 3, Autumn 2007, pp. 158-169). On the other hand, Wnt5a, a typical non-canonical Wnt member, has been shown to promote osteogenesis in vitro (Arnsdorf, E. J., et al., PLoS ONE, April 2009, Vol. 4, Issue 4, e5388, pp. 1-10; Baksh, D., et al., J. Cell. Physiol., 2007, 212: 817-826; J. Cell. Biochem., 2007, 101: 1109-1124). Since Wnt3a promotes MSC proliferation during early osteogenesis, it is thought likely that canonical Wnt signaling functions in the initiation of early osteogenic commitment by increasing the number of osteoprecursors in the stem cell compartment, while non-canonical Wnt drives the progression of osteoprecursors to mature functional osteoblasts.
Soluble Factors
Hepatocyte Growth Factor/Scatter Factor (HGF/SF)
Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that promotes mitogenesis, migration, invasion and morphogenesis (Jian, W. G. and S. Hiscox, Histol. Histopathol. 2: 537-555 (1997). HGF/SF signaling modulates integrin function by promoting aggregation and cell adhesion. Morphogenic responses to HGF/SF are dependent on adhesive events. See Matsumoto, K. et al, Cancer Metastasis Rev. 14: 205-217 (1995). HGF/SF-induced effects occur via signaling of the MET tyrosine kinase receptor following ligand binding, which leads to enhanced integrin-mediated B cell and lymphoma cell adhesion. Galimi, F. et al, Stem Cells 2: 22-30 (1993); Van der Voort, R. et al., J. Exp. Med. 185: 2121-31 (1997); Weimar, I. S. et al., Blood 89: 990-1000 (1997).
Tumor Growth Factor (Also Known as Transforming Growth Factor)
The TGF-β1 superfamily of structurally related peptides includes the TGF-β isoforms, β1, β2, β3, and β5, the activins and the bone morphogenetic proteins (BMPs). TGF-β-like factors are a multifunctional set of conserved growth and differentiation factors that control biological processes such as embryogenesis, organogenesis, morphogenesis of tissues like bone and cartilage, vasculogenesis, wound repair and angiogenesis, hematopoiesis, and immune regulation. Signaling by ligands of the TGF-β superfamily is mediated by a high affinity, ligand-induced, heteromeric complex consisting of related Ser/Thr kinase receptors divided into two subfamilies, type I and type II. The type II receptor transphosphorylates and activates the type I receptor in a Gly/Ser-rich region. The type I receptor in turn phosphorylates and transduces signals to a novel family of recently identified downstream targets, termed Smads.
Osteoprotegerin and RANKL
The molecules osteoprotegerin (OPG) and Receptor activator of NF-κB (RANKL) play a role in the communication between osteoclasts and osteoblasts and are members of a ligand-receptor system that directly regulates osteoclast differentiation and bone resorption. Grimaud, E. et al, Am J. Pathol. 2021-2031 (2993). RANKL has been shown to both activate mature osteoclasts and mediate osteoclastogenesis in the presence of M-CSF, i.e., RANKL is essential for osteoclast differentiation via its receptor RANK located on the osteoclast membrane. OPG is a soluble decoy receptor that prevents RANKL from binding to and activating RANK. It also inhibits the development of osteoclasts and down-regulates the RANKL signaling through RANK. RANKL and OPG have been detected in bone pathological situations where osteolysis occurred. The RANKL/OPG ratio is increased and correlated with markers of bone resorption, osteolytic lesions, and markers of disease activity in multiple myeloma. Id.
Macrophage Colony-Stimulating Factor (M-CSF)
Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is involved in the proliferation, differentiation, and survival of monocytes, macrophages, and bone marrow progenitor cells. Stanley E R, Berg K L, Einstein D B, Lee P S, Pixley F J, Wang Y, Yeung Y G, Mol. Reprod. Dev. 46 (1): 4-10 (1997).
Macrophage inflammatory protein 1-alpha (MIP1α) is a member of the C-C subrfamily of chemokines, a large superfamily of low-molecular weight, inducible proteins that exhibits a variety of proinflammatory activities in vitro. The C-C chemokines generally are chemotactic for cells of the monocyte lineage and lymphocytes. In addition to its proinflammatory activities, MIP-alpha inhibits the proliferation of hematopoietic stem cells in vitro and in vivo. Cook, D. N., J. Leukocyte Biol. 59(1): 61-66 (1996).
Sclerostin
Sclerostin, a protein expressed by osteocytes, downregulates osteoblastic bone formation by interfering with Wnt signaling.
Osteogenesis or Ossification
Osteogenesis or ossification is a process by which the bones are formed. There are three distinct lineages that generate the skeleton. The somites generate the axial skeleton, the lateral plate mesoderm generates the limb skeleton, and the cranial neural crest gives rise to the branchial arch, craniofacial bones, and cartilage. There are two major modes of bone formation, or osteogenesis, and both involve the transformation of a preexisting mesenchymal tissue into bone tissue. The direct conversion of mesenchymal tissue into bone is called intramembranous ossification. The process by which mesenchymal cells differentiate into cartilage, which is later replaced by bone cells is called endochondral ossification.
Intramembranous Ossification
Intramembraneous ossification is the characteristic way in which the flat bones of the scapula, the skull and the turtle shell are formed. In intramembraneous ossification, bones develop sheets of fibrous connective tissue. During intramembranous ossification in the skull, neural crest-derived mesenchymal cells proliferate and condense into compact nodules. Some of these cells develop into capillaries; others change their shape to become osteoblasts, committed bone precursor cells. The osteoblasts secrete a collagen-proteoglycan matrix that is able to bind calcium salts. Through this binding, the prebone (osteoid) matrix becomes calcified. In most cases, osteoblasts are separated from the region of calcification by a layer of the osteoid matrix they secrete. Occasionally, osteoblasts become trapped in the calcified matrix and become osteocytes. As calcification proceeds, bony spicules radiate out from the region where ossification began, the entire region of calcified spicules becomes surrounded by compact mesenchymal cells that form the periosteum, and the cells on the inner surface of the periosteum also become osteoblasts and deposit osteoid matrix parallel to that of the existing spicules. In this manner, many layers of bone are formed.
Intramembraneous ossification is characterized by invasion of capillaries into the mesenchymal zone, and the emergence and differentiation of mesenchymal cells into mature osteoblasts, which constitutively deposit bone matrix leading to the formation of bone spicules, which grow and develop, eventually fusing with other spicules to form trabeculae. As the trabeculae increase in size and number they become interconnected forming woven bone (a disorganized weak structure with a high proportion of osteocytes), which eventually is replaced by more organized, stronger, lamellar bone.
The molecular mechanism of intramembranous ossification involves bone morphogenetic proteins (BMPs) and the activation of a transcription factor called CBFA1. Bone morphogenetic proteins, for example, BMP2, BMP4, and BMP7, from the head epidermis are thought to instruct the neural crest-derived mesenchymal cells to become bone cells directly. BMPs activate the Cbfa1 gene in mesenchymal cells. The CBFA1 transcription factor is known to transform mesenchymal cells into osteoblasts. Studies have shown that the mRNA for mouse CBFA1 is largely restricted to the mesenchymal condensations that form bone, and is limited to the osteoblast lineage. CBFA1 is known to activate the genes for osteocalcin, osteopontin, and other bone-specific extracellular matrix proteins.
Endochondral Ossification (Intracartilaginous Ossification)
Endochondral ossification, which involves the in vivo formation of cartilage tissue from aggregated mesenchymal cells, and the subsequent replacement of cartilage tissue by bone, can be divided into five stages. The skeletal components of the vertebral column, the pelvis, and the limbs are first formed of cartilage and later become bone.
First, the mesenchymal cells are committed to become cartilage cells. This commitment is caused by paracrine factors that induce the nearby mesodermal cells to express two transcription factors, Pax1 and Scleraxis. These transcription factors are known to activate cartilage-specific genes. For example, Scleraxis is expressed in the mesenchyme from the sclerotome, in the facial mesenchyme that forms cartilaginous precursors to bone, and in the limb mesenchyme.
During the second phase of endochondral ossification, the committed mesenchyme cells condense into compact nodules and differentiate into chondrocytes (cartilage cells that produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans). Studies have shown that N-cadherin is important in the initiation of these condensations, and N-CAM is important for maintaining them. In humans, the SOX9 gene, which encodes a DNA-binding protein, is expressed in the precartilaginous condensations.
During the third phase of endochondral ossification, the chondrocytes proliferate rapidly to form the model for bone. As they divide, the chondrocytes secrete a cartilage-specific extracellular matrix.
In the fourth phase, the chondrocytes stop dividing and increase their volume dramatically, becoming hypertrophic chondrocytes. These large chondrocytes alter the matrix they produce (by adding collagen X and more fibronectin) to enable it to become mineralized by calcium carbonate.
The fifth phase involves the invasion of the cartilage model by blood vessels. The hypertrophic chondrocytes die by apoptosis, and this space becomes bone marrow. As the cartilage cells die, a group of cells that have surrounded the cartilage model differentiate into osteoblasts, which begin forming bone matrix on the partially degraded cartilage. Eventually, all the cartilage is replaced by bone. Thus, the cartilage tissue serves as a model for the bone that follows.
The replacement of chondrocytes by bone cells is dependent on the mineralization of the extracellular matrix. A number of events lead to the hypertrophy and mineralization of the chondrocytes, including an initial switch from aerobic to anaerobic respiration, which alters their cell metabolism and mitochondrial energy potential. Hypertrophic chondrocytes secrete numerous small membrane-bound vesicles into the extracellular matrix. These vesicles contain enzymes that are active in the generation of calcium and phosphate ions and initiate the mineralization process within the cartilaginous matrix. The hypertrophic chondrocytes, their metabolism and mitochondrial membranes altered, then die by apoptosis.
In the long bones of many mammals (including humans), endochondral ossification spreads outward in both directions from the center of the bone. As the ossification front nears the ends of the cartilage model, the chondrocytes near the ossification front proliferate prior to undergoing hypertrophy, pushing out the cartilaginous ends of the bone. The cartilaginous areas at the ends of the long bones are called epiphyseal growth plates. These plates contain three regions: a region of chondrocyte proliferation, a region of mature chondrocytes, and a region of hypertrophic chondrocytes. As the inner cartilage hypertrophies and the ossification front extends farther outward, the remaining cartilage in the epiphyseal growth plate proliferates. As long as the epiphyseal growth plates are able to produce chondrocytes, the bone continues to grow.
Bone Remodeling
Bone constantly is broken down by osteoclasts and re-formed by osteoblasts in the adult. This process of renewal is known as bone remodeling. The balance in this dynamic process shifts as people grow older: in youth, it favors the formation of bone, but in old age, it favors resorption.
As new bone material is added peripherally from the internal surface of the periosteum, there is a hollowing out of the internal region to form the bone marrow cavity. This destruction of bone tissue is due to osteoclasts that enter the bone through the blood vessels. Osteoclasts dissolve both the inorganic and the protein portions of the bone matrix. Each osteoclast extends numerous cellular processes into the matrix and pumps out hydrogen ions onto the surrounding material, thereby acidifying and solubilizing it. The blood vessels also import the blood-forming cells that will reside in the marrow for the duration of the organism's life.
The number and activity of osteoclasts must be tightly regulated. If there are too many active osteoclasts, too much bone will be dissolved, and osteoporosis will result. Conversely, if not enough osteoclasts are produced, the bones are not hollowed out for the marrow, and osteopetrosis (known as stone bone disease, a disorder whereby the bones harden and become denser) will result.
Lymphocytes and the Immune Response
Multicellular organisms have developed two defense mechanisms to fight infection by pathogens: innate and adaptive immune responses. Innate immune responses are triggered immediately after infection and are independent of the host's prior exposure to the pathogen. Adaptive immune responses operate later in an infection and are highly specific for the pathogen that triggered them. The function of adaptive immune responses is to destroy the invading pathogens and any toxic molecules they produce. (“Chapter 24: The adaptive immune system,” Molecular Biology of the Cell, Alberts, B. et al., Garland Science, N Y, 2002).
The immune system consists of a wide range of distinct cell types, amongst which white blood cells called lymphocytes play a central role in determining immune specificity. Other cells, such as monocytes, macrophages, dendritic cells, Langerhans' cells, natural killer (NK) cells, mast cells, basophils, and other members of the myeloid lineage of cells, interact with the lymphocytes and play critical functions in antigen presentation and mediation of immunologic functions. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
Lymphocytes are found in central lymphoid organs, the thymus, and bone marrow, where they undergo developmental steps that enable them to orchestrate immune responses. A large portion of lymphocytes and macrophages comprise a recirculating pool of cells found in the blood and lymph, providing the means to deliver immunocompetent cells to localized sites in need. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
Lymphocytes are specialized cells, committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence on the lymphocyte's surface of receptors that are specific for specific determinants or epitopes on the antigen. Each lymphocyte possesses a population of cell-surface receptors, all of which have identical combining regions. One set of lymphocyte, referenced to as a “clone” differs from another in the structure of the combining region of its receptors, and thus differs in the epitopes being recognized. The ability of an organism to respond to any nonself antigen is achieved by large number of different clones of lymphocytes, each bearing receptors specific for a distinct epitope. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
The adaptive immune system is composed of millions of lymphocyte clones. The diversity of lymphocytes is such that even a single antigenic determinant is likely to activate many clones, each of which produces an antigen-binding site with its own characteristic affinity for the determinant. Molec. Biol. Of the Cell, 1369). When many clones are activated, such responses are said to be polyclonal; when only a few clones are activated, the response is said to be oligoclonal, and when the response involves only a single B or T cell clone, it is said to be monoclonal.
There are two broad classes of adaptive immune responses that are carried out by different classes of lymphocytes: antibody responses mediated by B-lymphocytes (or B-cells); and cell-mediated immune responses carried out by T-lymphocytes (or T-cells). B-cells are bone-marrow-derived and are precursors of immunoglobulin- (Ig-) or antibody-expressing cells while T-cells are thymus-derived. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
Primary immune responses are initiated by the encounter of an individual with a foreign antigenic substance, generally an infectious microorganism. The infected individual responds with the production of immunoglobulin (Ig) molecules specific for the antigenic determinants of the immunogen and with the expansion and differentiation of antigen-specific regulatory and effector T-lymphocytes. The latter include both T-cells that secrete cytokines as well as natural killer T-cells that are capable of lysing the cell. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
As a consequence of the initial response, the immunized individual develops a state of immunologic memory. If the same (or closely related) microorganism or foreign object is encountered again, a secondary response is triggered. This generally consists of an antibody response that is more rapid and greater in magnitude than the primary (initial) response and is more effective in clearing the microbe from the body. A similar and more effective T-cell response then follows. The initial response often creates a state of immunity such that the individual is protected against a second infection, which forms the basis for immunizations. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
The immune response is highly specific. Primary immunization with a given microorganism evokes antibodies and T-cells that are specific for the antigenic determinants or epitopes found on that microorganism but that usually fail to recognize (or recognize only poorly) antigenic determinants of unrelated microbes. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
B-Lymphocytes:
B lymphocytes are a population of cells that express clonally diverse cell surface immunoglobulin (Ig) receptors recognizing specific antigenic epitopes.
B-lymphocytes are derived from hematopoietic stem cells by a complex set of differentiation events. The molecular events through which committed early members of the B lineage develop into mature B lymphocytes occur in fetal liver, and in adult life occur principally in the bone marrow. Interaction with specialized stromal cells and their products, including cytokines, such as interleukin IL-7, are critical to the normal regulation of this process. Tucker W. LeBien and Thomas F. Tedder, How they develop and function, Blood 112 (5): 1570-80 (2008). The phenotype of B cells generated with fetal liver is distinct from that using comparable precursors isolated from adult bone marrow. Richard R. Hardy and Kyoko Hayakawa, B Cell Development Pathways, Ann. Rev. Immunol. 19: 595-621 (2001).
Early B-cell development is characterized by the ordered rearrangement of Ig H and L chain loci, and Ig proteins themselves play an active role in regulating B-cell development.
Pre-B cells arise from progenitor (pro-B) cells that express neither the pre-B cell receptor (pre-BCR) or surface immunoglobulin (Ig).
Plasma cells, the critical immune effector cells dedicated to secretion of antigen-specific immunoglobulin (Ig) develop at three distinct stages of antigen-driven B cell development. Short-lived plasma cells emerge in response to both T-independent and T-dependent antigens. TD antigens also induce a germinal center (GC) pathway involving somatic hypermutation, affinity maturation, and production of memory B cells and long-lived PCs. Post-GC PCs have extended half-lives, produce high affinity antibody, and reside preferentially in the bone marrow. Memory B cells rapidly expand and differentiate into PCs in response to antigen challenge. Shapiro-Shelef, et al, Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells, Immunity 19: 607-20 (2003)
Antigen-induced B-cell activation and differentiation in secondary lymphoid tissues are mediated by dynamic changes in gene expression that give rise to the germinal center (GC) reaction (see section on B-cell maturation). Tucker W. LeBien and Thomas F. Tedder, How they develop and function, Blood 112 (5): 1570-80 (2008). The GC reaction is characterized by clonal expansion, class switch recombination (CSR) at the IgH locus, somatic hypermutation (SHM) of VH genes, and selection for increased affinity of a BCR for its unique antigenic epitope through affinity maturation.
Lymphocyte development requires the concerted action of a network of cytokines and transcription factors that positively and negatively regulate gene expression. Marrow stromal cell-derived interleukin-7 (IL-7) is a nonredundant cytokine for murine B-cell development that promotes V to DJ rearrangement and transmits survival/proliferation signals.
FLT3-ligand and TSLP play important roles in fetal B-cell development.
The cytokine(s) that regulate human B-cell development are not as well understood, and the cytokine (or cytokines) that promote marrow B-cell development at all stages of human life remains unknown.
At least 10 distinct transcription factors regulate the early stages of B-cell development, with E2A, EBF, and Pax5 being particularly important in promoting B-lineage commitment and differentiation.
Pax5, originally characterized by its capacity to bind to promoter sequences in Ig loci, may be the most multifunctional transcription factor for B cells. Pax5-deficient pro-B cells harbor the capacity to adapt non-B-lineage fates and develop into other hematopoietic lineages (Nutt S L, Heavey B, Rolink A G, Busslinger M., Nature. 1999; 401:556-562). Pax5 also regulates expression of at least 170 genes, a significant number of them important for B-cell signaling, adhesion, and migration of mature B cells (Cobaleda C, Schebesta A, Delogu A, Busslinger M., Nat Immunol. 2007; 8: 463-470). Conditional Pax5 deletion in mature murine B cells can result in dedifferentiation to an uncommitted hematopoietic progenitor and subsequent differentiation into T-lineage cells under certain conditions (Cobaleda C, Jochum W, Busslinger M., Nature. 2007; 449:473-477).
B lymphocyte induced maturation protein (Blimp-1), a transcriptional repressor, a 98 kDa protein containing five zinc finger motifs, has been implicated in plasma cell differentiation, and is required for the complete development of the pre-plasma memory B cell compartment. Shapiro-Shelef, et al, Blilmp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells, Immunity 19: 607-20 (2003).
B Cell Specific Cell Surface Molecules:
Table 1 shows Cell surface CD molecules that are preferentially expressed by B cells. Tucker W. LeBien and Thomas F. Tedder, How they develop and function, Blood 112 (5): 1570-80 (2008):
TABLE 1NameOriginal nameCellular ReactivityStructureCD19B4Pan-B cell, follicularIg superfamilydendritic cellsCD20B1Mature B cellsMS4A familyCD21B2, HB-5Mature B cells,ComplementFDCsreceptor familyCD22BL-CAM, Lyb-8Mature B cellsIg superfamilyCD23FcεRIIActivated B cells,C-type lectinFDCs, othersCD24BA-1, HB-6Pen-B cell,GPI anchoredgranulocytes,epithelial cellsCD40Bp50B cells, epithelialTNF receptorcells, FDCs, othersCD72Lyb-2Pam-B cellC-type lectinCD79 a, bIgε,βSurface Ig+ B cellsIg superfamily
CD19 is expressed by essentially all B-lineage cells and regulates intracellular signal transduction by amplifying Src-family kinase activity. CD20 is a mature B cell-specific molecule that functions as a membrane-embedded Ca2+ channel. Importantly, ritixumab, the first mAb approved by the Food and Drug Administration (FDA) for clinical use in cancer therapy (eg, follicular lymphoma), is a chimeric CD20 mAb.
CD21 is the C3d and Epstein-Barr virus receptor that interacts with CD19 to generate transmembrane signals and inform the B cell of inflammatory responses within microenvironments.
CD22 functions as a mammalian lectin for α2,6-linked sialic acid that regulates follicular B-cell survival and negatively regulates signaling.
CD23 is a low-affinity receptor for IgE expressed on activated B cells that influences IgE production.
CD24 was among the first pan-B-cell molecules to be identified, but this unique GPI-anchored glycoprotein's function remains unknown.
CD40 serves as a critical survival factor for GC B cells and is the ligand for CD154 expressed by T cells.
CD72 functions as a negative regulator of signal transduction and as the B-cell ligand for Semaphorin 4D (CD100).
There may be other unidentified molecules preferentially expressed by B cells, but the cell surface landscape is likely dominated by molecules shared with multiple leukocyte lineages.
B-Cell Maturation and Subset Development
Outside the marrow, B cells are morphologically homogenous, but their cell surface phenotypes, anatomic localization, and functional properties reveal still-unfolding complexities. Immature B cells exiting the marrow acquire cell surface IgD as well as CD21 and CD22, with functionally important density changes in other receptors. Immature B cells are also referred to as “transitional” (T1 and T2) based on their phenotypes and ontogeny, and have been characterized primarily in the mouse (Chung J B, Silverman M, Monroe J G., Trends Immunol. 2003; 24:343-349). Immature B cells respond to T cell-independent type 1 antigens such as lipopolysaccharides, which elicit rapid antibody responses in the absence of MHC class II-restricted T-cell help (Coutinho A, Moller G., Adv Immunol. 1975; 21:113-236). The majority of mature B cells outside of the gut associated lymphoid tissue (GALT) reside within lymphoid follicles of the spleen and lymph nodes, where they encounter and respond to T cell-dependent foreign antigens bound to follicular dendritic cells (DCs), proliferate, and either differentiate into plasma cells or enter GC reactions.
Germinal centers (GCs), which refers to sites within lymphoid tissue that are more active in lymphocyte proliferation than are other parts of the lymphoid tissue, containing rapidly proliferating cells (ie, centroblasts) are the main site for high-affinity antibody-secreting plasma cell and memory B-cell generatior (Jacob J, Kelsoe G, Rajewsky K, Weiss U., Nature. 1991; 354:389-392). Within GCs, somatic hypermutation (SHM) and purifying selection produce the higher affinity B-cell clones that form the memory compartments of humoral immunity (Jacob J, Kelsoe G, Rajewsky K, Weiss U., Nature. 1991; 354:389-392; Kelsoe G., Immunity. 1996; 4:107-111). Affinity maturation in GCs does not represent an intrinsic requirement for BCR signal strength but rather a local, Darwinian competition. The dynamics of lymphocyte entry into follicles and their selection for migration into and within GCs represents a complex ballet of molecular interactions orchestrated by chemotactic gradients and B-cell receptor (BCR) engagement that is only now being elucidated (Allen C D, Okada T, Cyster J G., Immunity. 2007; 27:190-202).
B-cell subsets with individualized functions such as B-1 and marginal zone (MZ, referring to the junction of the lymphoid tissue of a lymphatic nodule with the surrounding non-lymphoid red pulp of the spleen) B cells have also been identified. Murine B-1 cells are a unique CD5+ B-cell subpopulation (Hayakawa K, Hardy R R, Parks D R, Herzenberg L A., J Exp Med. 1983; 157:202-218) distinguished from conventional B (B-2) cells by their phenotype, anatomic localization, self-renewing capacity, and production of natural antibodies (Hardy R R, Hayakawa K., Annu Rev Immunol. 2001; 19:595-621). Peritoneal B-1 cells are further subdivided into the B-1a (CD5+) and B-1b (CD5−) subsets. Their origins, and whether they derive from the same or distinct progenitors compared with B-2 cells, have been controversial (Dorshkind K, Montecino-Rodriguez E., Nat Rev Immunol. 2007; 7:213-219). However, a B-1 progenitor that appears distinct from a B-lineage progenitor that develops primarily into the B-2 population has been identified in murine fetal marrow, and to a lesser degree in adult marrow (Montecino-Rodriguez E, Leathers H, Dorshkind K., Nat Immunol. 2006; 7:293-301). B-1a cells and their natural antibody products provide innate protection against bacterial infections in naive hosts, while B-1b cells function independently as the primary source of long-term adaptive antibody responses to polysaccharides and other T cell-independent type 2 antigens during infection (Montecino-Rodriguez E, Leathers H, Dorshkind K., Nat Immunol. 2006; 7:293-301). The function and potential subpopulation status of human B-1 cells is less understood (Dorshkind K, Montecino-Rodriguez E., Nat Rev Immunol. 2007; 7:213-219). MZ B cells are a unique population of murine splenic B cells with attributes of naive and memory B cells (Pillai S, Cariappa A, Moran S T., Annu Rev Immunol. 2005; 23:161-196), and constitute a first line of defense against blood-borne encapsulated bacteria. Uncertainty regarding the identity of human MZ B cells partially reflects the fact that the microscopic anatomy of the human splenic MZ differs from rodents (Steiniger B, Timphus E M, Barth P J., Histochem Cell Biol. 2006; 126:641-648) Likewise, the microscopic anatomy of human follicular mantle zones is not recapitulated in mouse spleen and lymph nodes.
The B1, MZ, and GC B-cell subsets all contribute to the circulating natural antibody pool, thymic-independent IgM antibody responses, and adaptive immunity by terminal differentiation into plasma cells, the effector cells of humoral immunity (Radbruch A, Muehlinghaus G, Luger E O, et al., Nat Rev Immunol. 2006; 6:741-750). Antigen activation of mature B cells leads initially to GC development, the transient generation of plasmablasts that secrete antibody while still dividing, and short-lived extrafollicular plasma cells that secrete antigen-specific germ line-encoded antibodies (FIG. 1). GC-derived memory B cells generated during the second week of primary antibody responses express mutated BCRs with enhanced affinities, the product of SHM. Memory B cells persist after antigen challenge, rapidly expand during secondary responses, and can terminally differentiate into antibody-secreting plasma cells. In a manner similar to the early stages of B-cell development in fetal liver and adult marrow, plasma cell development is tightly regulated by a panoply of transcription factors, most notably Bcl-6 and BLIMP-1 (Shapiro-Shelef M, Calame K., Nat Rev Immunol. 2005; 5:230-242).
Persistent antigen-specific antibody titers derive primarily from long-lived plasma cells (Radbruch A, Muehlinghaus G, Luger E O, et al., Nat Rev Immunol. 2006; 6:741-750). Primary and secondary immune responses generate separate pools of long-lived plasma cells in the spleen, which migrate to the marrow where they occupy essential survival niches and can persist for the life of the animal without the need for self-replenishment or turnover ((Radbruch A, Muehlinghaus G, Luger E O, et al., Nat Rev Immunol. 2006; 6:741-750; McHeyzer-Williams L J, McHeyzer-Williams M G., Annu Rev Immunol. 2005; 23:487-513). The marrow plasma cell pool does not require ongoing contributions from the memory B-cell pool for its maintenance, but when depleted, plasma cells are replenished from the pool of memory B cells (Dilillo D J, Hamaguchi Y, Ueda Y, et al., J Immunol. 2008; 180:361-371). Thereby, persisting antigen, cytokines, or Toll-like receptor signals may drive the memory B-cell pool to chronically differentiate into long-lived plasma cells for long-lived antibody production.
In addition to their essential role in humoral immunity, B cells also mediate/regulate many other functions essential for immune homeostasis (FIG. 2). Of major importance, B cells are required for the initiation of T-cell immune responses, as first demonstrated in mice depleted of B cells at birth using anti-IgM antiserum (Ron Y, De Baetselier P, Gordon J, Feldman M, Segal S., Eur J Immunol. 1981; 11:964-968). However, this has not been without controversy as an absence of B cells impairs CD4 T-cell priming in some studies, but not others. Nonetheless, antigen-specific interactions between B and T cells may require the antigen to be first internalized by the BCR, processed, and then presented in an MHC-restricted manner to T cells (Ron Y, Sprent J., J Immunol. 1987; 138:2848-2856; Janeway C A, Ron J, Jr, Katz M E., J Immunol. 1987; 138:1051-1055; Lanzavecchia A., Nature. 1985; 314:537-539).
B-Cell Abnormalities:
The normal B-cell developmental stages have malignant counterparts that reflect the expansion of a dominant subclone leading to development of leukemia and lymphoma.
For example, non-T, non-B ALL is a malignancy of B-cell precursors (Korsmeyer S J, Arnold A, Bakhshi A, et al., J Clin Invest. 1983; 71:301-313). The antiapoptotic Bcl-2 gene was discovered as the translocation partner with the IgH locus in the t(14; 18)(q32; q21); frequently occurring in follicular lymphoma (Tsujimoto Y, Finger L R, Yunis J, Nowell P C, Croce C M., Science. 1984; 226:1097-1099). A substantial number of cases of diffuse large B-cell lymphoma exhibit dysregulated expression of the transcriptional repressor Bcl-6 (Ye B H, Lista F, Lo Coco F, et al., Science. 1993; 262:747-750). The Hodgkin/Reed-Sternberg cell in Hodgkin lymphoma, is of B-lymphocyte origin based on the demonstration of clonal Ig gene rearrangements (Kuppers R, Rajewsky K, Zhao M, et al., Proc Natl Acad Sci USA. 1994; 91: 10962-10966).
The monoclonal gammopathies (paraproteinemias or dysproteinemias) are a group of disorders characterized by the proliferation of a single clone of plasma cells which produces an immunologically homogeneous protein commonly referred to as a paraprotein or monoclonal protein (M-protein, where the “M” stands for monoclonal). Each serum M-protein consists of two heavy polypeptide chains of the same class designated by a capital letter and a corresponding Greek letters: Gamma (γ) in IgG, Alpha (α) in IgA, Mu (μ) in IgM, Delta (δ) in IgD, Epsilon (ε) in IgE. For example, basophils in IgE myeloma are characterized by a higher expression of high affinity IgE receptor relative to normal controls.
Multiple Myeloma
Multiple myeloma (MM), a B cell malignancy characterized by the accumulation of plasma cells in the BM and the secretion of large amounts of monoclonal antibodies that ultimately causes bone lesions, hypercalcaemia, renal disease, anemia, and immunodeficiency (Raab M S, Podar K, Breitkreutz I, Richardson P G, Anderson K C., Lancet 2009; 374:324-39), is the second most frequent blood disease in the United States affecting 7.1 per 100,000 men and 4.6 per 100,000 women.
MM is characterized by monoclonal proliferation of malignant plasma cells (PCs) in the bone marrow (BM), the presence of high levels of monoclonal serum antibody, the development of osteolytic bone lesions, and the induction of angiogenesis, neutropenia, amyloidosis, and hypercalcemia (Vanderkerken K, Asosingh K, Croucher P, Van Camp B., Immunol Rev 2003; 194:196-206; Raab M S, Podar K, Breitkreutz I, Richardson P G, Anderson K C., Lancet 2009; 374:324-39). MM is seen as a multistep transformation process. G. Pratt., Molecular Aspects of multiple myeloma, J. Clin. Pathol: Molec. Pathol. 55: 273-83 (2002). Although little is known about the immortalizing and initial transforming events, the initial event is thought to be the immortalization of a plasma cell to form a clone, which may be quiescent, non-accumulating and not cause end organ damage due to accumulation of plasma cells within the bone marrow (MGUS). Smouldering MM (SMM) also has no detectable end-organ damage, but differs from MGUS by having a serum mIg level higher than 3 g/dl or a BM PC content of more than 10% and an average rate of progression to symptomatic MM of 10% per year. Currently there are no tests that measure phenotypic or genotypic markers on tumor cells that predict progression. W. Michael Kuehl and P. Leif Bergsagel, Molecular pathogenesis of multiple myeloma and its premalignant precursor, J. Clin. Invest. 122 (10): 3456-63 (2012). An abnormal immunophenotype distinguishes healthy plasma cells (PCs) from tumor cells. Healthy BM PCs are CD38+CD138+CD19+CD45+CD56−. Id. Although MM tumor cells also are CD38+CD138+, 90% are CD19−, 99% are CD45− or CD45 lo, and 70% are CD56+. Id.
The prognosis and treatment of this disease has greatly evolved over the past decade due to the incorporation of new agents that act as immunomodulators and proteosome inhibitors. Despite recent progress with a number of novel treatments (Raab M S, Podar K, Breitkreutz I, Richardson P G, Anderson K C., Lancet 2009; 374:324-39; Schwartz R N, Vozniak M., J Manag Care Pharm 2008; 14:12-9), patients only experience somewhat longer periods of remission. Because of the development of drug resistance or relapse, MM is an incurable disease (Schwartz R N, Vozniak M., J Manag Care Pharm 2008; 14:12-9; Kyle R A., Blood 2008; 111:4417-8), with a median survival time of 3-4 years.
Disease management is currently tailored based on the patient's co-morbidity factors and stage of disease (for a complete list of treatments and their implementation, see Raab M S, Podar K, Breitkreutz I, Richardson P G, Anderson K C., Lancet 2009; 374:324-39 and Schwartz R N, Vozniak M., J Manag Care Pharm 2008; 14:12-9).
Staging of Myeloma
While multiple myeloma may be staged using the Durie-Salmon system, its value is becoming limited because of newer diagnostic methods. The International Staging System for Multiple Myeloma relies mainly on levels of albumin and beta-2-microglobulin in the blood. Other factors that may be important are kidney function, platelet count and the patient's age. [www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-staging, last revised Feb. 12, 2013]
The Durie-Salmon staging system is based on 4 factors:
The amount of abnormal monoclonal immunoglobulin in the blood or urine: Large amounts of monoclonal immunoglobulin indicate that many malignant plasma cells are present and are producing that abnormal protein.
The amount of calcium in the blood: High blood calcium levels can be related to advanced bone damage. Because bone normally contains lots of calcium, bone destruction releases calcium into the blood.
The severity of bone damage based on x-rays: Multiple areas of bone damage seen on x-rays indicate an advanced stage of multiple myeloma.
The amount of hemoglobin in the blood: Hemoglobin carries oxygen in red blood cells. Low hemoglobin levels mean that the patient is anemic; it can indicate that the myeloma cells occupy much of the bone marrow and that not enough space is left for the normal marrow cells to make enough red blood cells.
This system uses these factors to divide myeloma into 3 stages. Stage I indicates the smallest amount of tumor, and stage III indicates the largest amount of tumor:
In Stage I, a relatively small number of myeloma cells are found. All of the following features must be present:
Hemoglobin level is only slightly below normal (still above 10 g/dL)
Bone x-rays appear normal or show only 1 area of bone damage
Calcium levels in the blood are normal (less than 12 mg/dL)
Only a relatively small amount of monoclonal immunoglobulin is in blood or urine
In Stage II, a moderate number of myeloma cells are present. Features are between stage I and stage III.
In Stage III, a large number of myeloma cells are found. One or more of the following features must be present:
Low hemoglobin level (below 8.5 g/dL)
High blood calcium level (above 12 mg/dL)
3 or more areas of bone destroyed by the cancer
Large amount of monoclonal immunoglobulin in blood or urine
The International Staging System divides myeloma into 3 stages based only on the serum beta-2 microglobulin and serum albumin levels.
In Stage I, serum beta-2 microglobulin is less than 3.5 (mg/L) and the albumin level is above 3.5 (g/L). Stage II is neither stage I nor III, meaning that either: The beta-2 microglobulin level is between 3.5 and 5.5 (with any albumin level), OR the albumin is below 3.5 while the beta-2 microglobulin is less than 3.5. In Stage III, Serum beta-2 microglobulin is greater than 5.5.
Factors other than stage that affect survival include kidney function (when the kidneys are damaged by the monoclonal immunoglobulin, blood creatinine levels rise, predicting a worse outlook); age (in the studies of the international staging system, older people with myeloma do not live as long); the myeloma labeling index (sometimes called the plasma cell labeling index), which, indicates how fast the cancer cells are growing; a high labeling index can predict a more rapid accumulation of cancer cells and a worse outlook; and chromosome studies, i.e., certain chromosome changes in the malignant cells can indicate a poorer outlook. For example, changes in chromosome 13 will lower a person's chances for survival. Another genetic abnormality that predicts a poor outcome is a translocation (meaning an exchange of material) from chromosomes 4 and 14.
Biological pharmacotherapy for the treatment of MM currently includes immunomodulatory agents, such as thalidomide or its analogue, lenalidomide, and bortezomib, a first-in-class proteosome inhibitor. Unfortunately, some side effects associated with these therapies such as peripheral neuropathy and thrombocytopenia (in the case of bortezomib) restrict dosing and duration of treatment (Raab M S, Podar K, Breitkreutz I, Richardson P G, Anderson K C., Lancet 2009; 374:324-39; Schwartz R N, Vozniak M., J Manag Care Pharm 2008; 14:12-9; Field-Smith A, Morgan G J, Davies F E., Ther Clin Risk Manag 2006; 2:271-9).
Despite significant advances in the implementation of these drugs, MM still remains a lethal disease for the vast majority of patients. Since MM is a disease characterized by multiple relapses, the order/sequencing of the different effective treatment options is crucial to the outcome of MM patients. In the frontline setting, the first remission is likely to be the period during which patients will enjoy the best quality of life. Thus, one goal is to achieve a first remission that is the longest possible by using the most effective treatment upfront. At relapse, the challenge is to select the optimal treatment for each patient while balancing efficacy and toxicity. The decision will depend on both disease- and patient-related factors (Mohty B, El-Cheikh J, Yakoub-Agha I, Avet-Loiseau H, Moreau P, Mohty M., Leukemia 2012; 26:73-85). Thus, having the capability of testing the efficacy of a potential therapy, prior to patient treatment, can have a major impact in the management of this disease.
As opposed to other hematological malignancies, MM as well as other cancers that metastasize to the BM strongly interact with the BM microenvironment, which is composed of endothelial cells, stromal cells, osteoclasts (OCL), osteoblasts (OSB), immune cells, fat cells and the extracellular matrix (ECM). These interactions, as illustrated in FIG. 1 (adapted from Roodman G D., Bone 2011; 48:135-40), are responsible for the specific homing in the BM, the proliferation and survival of the MM cells, the resistance of MM cells to drug treatment, and the development of osteolysis, immunodeficiency, and anemia (Dvorak H F, Weaver V M, Tlsty T D, Bergers G., J Surg Oncol 2011; 103:468-74; De Raeve H R, Vanderkerken K., Histol Histopathol 2005; 20:1227-50; Fowler J A, Edwards C M, Croucher P I., Bone 2011; 48:121-8; Fowler J A, Mundy G R, Lwin S T, Edwards C M., Cancer Res 2012; Roodman G D., J Bone Miner Res 2002; 17:1921-5).
The Bone Marrow Niche and MM Progression
The BM niche plays a key role in MM-related bone disease. A complex interaction with the BM microenvironment in areas adjacent to tumor foci, characterized by activation of osteoclasts and suppression of osteoblasts, leads to lytic bone disease. W. Michael Kuehl and P. Leif Bergsagel, Molecular pathogenesis of multiple myeloma and its premalignant precursor, J. Clin. Invest. 122 (10): 3456-63 (2012); Shmuel Yaccoby, Advances in the understanding of myeloma bone disease and tumour growth, Br. J. Haematol. 149 (3): 311-321 (2010). Thus, although the MM microenvironment is highly complex, it is understood that suppression of OSB activity plays a key role in the bone destructive process as well as progression of the tumor burden (Roodman G D., Bone 2011; 48:135-40). Treatments that target both the bone microenvironment as well as the tumor, such as bortezomib and immunomodulatory drugs, have been more effective than prior therapies for MM and have dramatically increased both progression-free survival and overall survival of patients.
MM cells closely interact with the BM microenvironment, also termed the cancer niche. The elements of the bone marrow niche can provide an optimal growth environment for multiple hematological malignancies including multiple myeloma (MM). MM cells convert the bone marrow into specialized neoplastic niche, which aids the growth and spreading of tumor cells by a complex interplay of cytokines, chemokines, proteolytic enzymes and adhesion molecules. Moreover, the MM BM microenvironment confers survival and chemoresistance of MM cells to current therapies.
Bone Marrow Stromal Cells (BMSCs)
Multiple myeloma (MM) cells adhere to BMSC and ECM. Tumor cells, such as MM cells, bind to ECM proteins, such as type I collagen and fibronectin via syndecan 1 and very late antigen 4 (VLA-4) on MM cells and to BMSC VCAM-1 via VLA-4 on MM cells. Adhesion of MM cells to BMSC activates many pathways resulting in upregulation of cell cycle regulating proteins and antiapoptotic proteins (Hideshima T, Bergsagel P L, Kuehl W M, Anderson K C., Blood. 2004; 104(3):607-618). The interaction between MM cells and BMSCs triggers NF-κB signaling pathway and interleukin-6 (IL-6) secretion in BMSCs. In turn, IL-6 enhances the production and secretion of VEGF by MM cells. The existence of this paracrine loop optimizes the BM milieu for MM tumor cell growth (Kumar S, Witzig T E, Timm M, et al., Leukemia. 2003; 17(10):2025-2031). BMSC-MM cell interaction is also mediated through Notch. The Notch-signaling pathways both in MM cells as well as in BMSC, promote the induction of IL-6, vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1) secretion and is associated with MM cell proliferation and survival (Radtke F, Raj K., Nature Reviews Cancer. 2003; 3(10):756-767; Nefedova Y, Cheng P, Alsina M, Dalton W S, Gabrilovich D I., Blood. 2004; 103(9):3503-3510). It has been shown that BMSC from MM patients expresses several proangiogenic molecules, such as VEGF, basic-fibroblast growth factor (bFGF), angiopoietin 1 (Ang-1), transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and interleukin-1 (IL-1) (Giuliani N, Storti P, Bolzoni M, Palma B D, Bonomini S., Cancer Microenvironment. 2011; 4(3):325-337). BMSCs from MM patients also have been shown to release exosomes, which are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo, mediated by specific miRNA (Roccaro A M, Sacco A, Azab A K, et al., Blood. 2011; 118, abstract 625 ASH Annual Meeting Abstracts).
Endothelial Cells and Angiogenesis
BM angiogenesis represents a constant hallmark of MM progression, partly driven by release of pro-angiogenic cytokines from the tumor plasma cells, BMSC, and osteoclasts, such as VEGF, bFGF, and metalloproteinases (MMPs). The adhesion between MM cells and BMSCs upregulates many cytokines with angiogenic activity, most notably VEGF and bFGF (Podar K, Anderson K C., Blood. 2005; 105(4):1383-1395). In MM cells, these pro-angiogenic factors may also be produced constitutively as a result of oncogene activation and/or genetic mutations (Rajkumar S V, Witzig T E., Cancer Treatment Reviews. 2000; 26(5):351-362). Evidence for the importance of angiogenesis in the pathogenesis of MM was obtained from BM samples from MM patients (Kumar S, Gertz M A, Dispenzieri A, et al., Bone Marrow Transplantation. 2004; 34(3):235-239). The level of BM angiogenesis, as assessed by grading and/or microvessel density (MVD), is increased in patients with active MM as compared to those with inactive disease or monoclonal gammopathy of undetermined significance (MGUS), a less advanced plasma cell disorder. Comparative gene expression profiling of multiple myeloma endothelial cells and MGUS endothelial cells has been performed in order to determine a genetic signature and to identify vascular mechanisms governing the malignant progression (Ria R, Todoerti K, Berardi S, et al., Clinical Cancer Research. 2009; 15(17):5369-5378). Twenty-two genes were found differentially expressed at relatively high stringency in MM endothelial cells compared with MGUS endothelial cells. Functional annotation revealed a role of these genes in the regulation of ECM formation and bone remodelling, cell adhesion, chemotaxis, angiogenesis, resistance to apoptosis, and cell-cycle regulation. The distinct endothelial cell gene expression profiles and vascular phenotypes detected may influence remodelling of the bone marrow microenvironment in patients with active multiple myeloma. Overall, these evidences suggest that EC presents with functional, genetic, and morphologic features indicating their ability to induce BM neovascularization, resulting in MM cell growth, and disease progression.
Osteoclasts
The usual balance between bone resorption and new bone formation is lost in many cases of MM, resulting in bone destruction and the development of osteolytic lesions (Bataille R, Chappard D, Marcelli C, et al., Journal of Clinical Oncology. 1989; 7(12):1909-1914). Bone destruction develops adjacent to MM cells, yet not in areas of normal bone marrow. There are several factors implicated in osteoclast activation, including receptor activator of NF-κB ligand (RANKL), macrophage inflammatory protein-1a (MIP-1a), interleukin-3 (IL-3), and IL-6 (Roodman G D., Leukemia. 2009; 23(3):435-441). RANK ligand is a member of the tumor necrosis factor (TNF) family and plays a major role in the increased osteoclastogenesis implicated in MM bone disease. RANK is a transmembrane signaling receptor expressed by osteoclast cells. MM cell binding to neighboring BMSC within the bone marrow results in increased RANKL expression. This leads to an increase in osteoclast activity through the binding of RANKL to its receptor, on osteoclast precursor cells, which further promotes their differentiation through NF-κB and JunN-terminal kinase pathway (Ehrlich L A, Roodman G D., Immunological Reviews. 2005; 208:252-266). RANKL is also involved in inhibition of osteoclast apoptosis. Blocking RANKL with a soluble form of RANK has been shown to modulate not only bone loss but also tumor burden in MM in vivo models (Yaccoby S, Pearse R N, Johnson C L, Barlogie B, Choi Y, Epstein J., British Journal of Haematology. 2002; 116(2):278-290). Moreover osteoclasts constitutively secrete proangiogenic factors osteopontin that enhanced vascular tubule formation (Tanaka Y, Abe M, Hiasa M, et al., Clinical Cancer Research. 2007; 13(3):816-823).
Osteoblasts in MM Progression
Osteoblasts are thought to contribute to MM pathogenesis by supporting MM cells growth and survival (Karadag A, Oyajobi B O, Apperley J F, Graham R, Russell G, Croucher P I., British Journal of Haematology. 2000; 108(2):383-390). This could potentially result from the ability of osteoblasts to secrete IL-6 in a co-culture system with MM cells, thus increasing IL-6 levels within the BM milieu and inducing MM plasma cell growth. Other mechanisms include the possible role of osteoblasts in stimulating MM cell survival by blocking TRAIL-mediated programmed MM cell death, by secreting osteoprotegerin (OPG), a receptor for both RANKL and TRAIL (Shipman C M, Croucher P I., Cancer Research. 2003; 63(5):912-916). In addition, suppression of osteoblast activity is responsible for both bone destructive process and progression of myeloma tumor burden. Several factors have been implicated in the suppression of osteoblast activity in MM, including DKK1 (Tian E, Zhan F, Walker R, et al., The New England Journal of Medicine. 2003; 349(26):2483-2494). DKK1 is a Wnt-signaling antagonist secreted by MM cells that inhibits osteoblast differentiation. DKK1 is significantly overexpressed in patients with MM who present with lytic bone lesions. Myeloma-derived DKK1 also disrupts Wnt-regulated OPG and RANKL production by osteoblasts. Studies have shown that blocking DKK1 and activating Wnt signaling prevents bone disease in MM and is associated with a reduction in tumor burden (Yaccoby S, Ling W, Zhan F, Walker R, Barlogie B, Shaughnessy J D., Jr., Blood. 2007; 109(5):2106-2111; Edwards C M, Edwards J R, Lwin S T, et al., Blood. 2008; 111(5):2833-2842; Fulciniti M, Tassone P, Hideshima T, et al., Blood. 2009; 114(2):371-379).
Many components of the microenvironment support the propagation of the MM cells through cell-cell adhesion and the release of growth factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) (Deleu S, Lemaire M, Arts J, et al., Leukemia 2009; 23:1894-903; Field-Smith A, Morgan G J, Davies F E., Ther Clin Risk Manag 2006; 2:271-9; D'Souza S, del Prete D, Jin S, et al. Blood 2011; 118:6871-80). Survival and drug resistance of malignant cells is associated with their ability to shape the local microenvironment, in part by disrupting the balance of pro- and anti-angiogenic factors through neovascularization (Otjacques E, Binsfeld M, Noel A, Beguin Y, Cataldo D, Caers J., Int J Hematol 2011; 94:505-18) and bone remodeling which leads to osteolysis (Raje N, Roodman G D., Clin Cancer Res 2011; 17:1278-86; Giuliani N, Rizzoli V, Roodman G D., Blood 2006; 108:3992-6; Lentzsch S, Ehrlich L A, Roodman G D., Hematol Oncol Clin North Am 2007; 21:1035-49, viii).
Unfortunately, primary MM tumor cells have been difficult to propagate ex vivo because they require a microenvironment hard to reproduce in vitro. MM cells grown in vitro therefore are very short lived and grow poorly outside their BM milieu and attempts to optimize their maintenance have been hampered by lack of known conditions that allow for their ex vivo survival (Zlei M, Egert S, Wider D, Ihorst G, Wasch R, Engelhardt M., Exp Hematol 2007; 35:1550-61). Aside from various xenograft models (Calimeri T, Battista E, Conforti F, et al., Leukemia 2011; 25:707-11; Yata K, Yaccoby S., Leukemia 2004; 18:1891-7; Yaccoby S, Johnson C L, Mahaffey S C, Wezeman M J, Barlogie B, Epstein J., Blood 2002; 100:4162-8; Bell E., Nature Reviews Immunology 2006; 6:87), only one group to date has reported on creating an in vitro model capable of supporting the proliferation and survival of MM cells (Kirshner J, Thulien K J, Martin L D, et al., Blood 2008; 112:2935-45). However, the macroscale static methodology that was employed has limited value as, inter alia, it fails to recapitulate the spatial and temporal characteristics of the complex tumor niche.
Recently, Lee et al described a three-dimensional (3D) tissue construct in which a multichannel microfluidic device was used to create mineralized 3D tissue-like structures by dynamic long-term culture of osteoblasts to evaluate efficacy of biomaterials aimed at accelerating orthopedic implant related wound healing while preventing bacterial infection. Development of osteoblasts into 3D tissue-like structures and how this development was influenced by interaction with the pathogen Staphylococcus epidermidis was studied in real-time. Lee, et al., Microfluidic approach to create three-dimensional tissue models for biofilm-related infection of orthopoedic implants, Tissue Engineering: Part C, 17 (1): 39-48 (2011); Lee, et al., Microfluidic 3D bone tissue model for high throughput evaluation of wound healing and infection-preventing biomaterials,” Biomaterials 33: 999-1006 (2012). It was observed that in the absence of bacteria, osteoblasts formed a confluent layer on the bottom channel surface, gradually migrated to the side and top surfaces, and formed calcified 3D nodular structures in 8 days.
This 3D biological construct now has been used to create a microfluidic 3D MM/bone tissue model, which provides a perfused microenvironment, facilitates the seeding of adherent and non-adherent BM cells, and accelerates reconstruction of the BM milieu. The model system better preserves the BM/MM interactions, and, from a clinical perspective, enables a physiologically relevant system that: 1) maximizes sample use by requiring very small amounts of patient BM cells (<1×106 cells) and plasma (<2 mL/culture/week) and 2) accelerates the evaluation of new therapeutics for the treatment of MM. Furthermore, because real-time monitoring of BM/MM cell developments and interactions are performed, the described model is useful to study and identify new mechanisms associated with the MM niche and tumor progression. For example, use of the microfluidic 3D MM/bone tissue model to evaluate effects of soluble factors secreted by MM cells on the maintenance of the microfluidic 3D bone tissue has been reported. Zhang, et al., Microfluidic 3D bone tissue model for multiple myeloma, 9th World Biomaterials Congress, Jun. 5, 2012.
In addition, although MM has been used as a model system, the conservation of the BM microenvironment from BM biospecimens has broader utility in the study of other blood cancers and solid tumors that reside or metastasize to the BM.